SCRIPT Reverse Transcriptase

Reverse Transcriptase with increased thermal stability

Cat. No. Amount Applications:

Extremely sensitive and highly specific RT-PCR, synthesis of highly
PCR-505S 20.000 units
structured and long cDNA fragments, DNA labelling.
PCR-505L 100.000 units
Description:
SCRIPT Reverse Transcriptase is a genetically engineered version
Unit Definition: One unit is defined as the amount of enzyme of M-MLV Reverse Transcriptase (M-MLV RT) with eliminated RNase
required to catalyze the incorporation of 1 nmol of dTTP into an H activity and increased thermal stability. The enzyme is a RNA-
acid-insoluble form in 10 minutes at 37 °C. directed DNA polymerase that synthesizes a complementary DNA
strand initiating from a primer using single-stranded RNA or DNA
For in vitro use only! as template. Its enhanced thermal stability in combination with
the deactivated RNase H activity results in an increased specificity,
higher cDNA yield and an improved efficiency for full length
Shipping: shipped on blue ice
cDNA synthesis compared with standard M-MLV RT. The enzyme
Storage Conditions: store at -20 °C is recommended for synthesis of cDNA from 100 bp up to 10 kb length.

Additional Storage Conditions: avoid freeze/thaw cycles Content:

Shelf Life: 12 months SCRIPT Reverse Transcriptase (red cap)
200 units/µl in 50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA,
Purity: free of endo- and exodeoxyribonucleases, phosphatases and 5 mM DTT, 0.1 % NP-40, 50 % glycerol (v/v), pH 8.0 (25°C)
ribonuclease
Form: liquid 5x SCRIPT RT Buffer complete (green cap)
250 mM Tris-HCl (pH 8.3), 500 mM KCl, 30 mM MgCl2 , 25 mM DTT
Concentration: 200 units/µl
dNTP Mix (white cap)
10 mM each dNTP (dATP, dCTP, dGTP, dTTP)

DTT stock solution (purple cap)

100 mM DTT

Recommended protocols for cDNA synthesis:

Sample denaturation is particularly recommended for RNA targets
that exhibit a high degree of secondary structure, for self- or
cross-complementary primers and for initial experiments with new
targets. For many standard combinations of RNA and primers heat
treatment may be omitted with no negative effect on results.

1a Assay set-up without sample denaturation

(standard RNA/primer combinations)
Assay preparation
Add the following components to a nuclease-free microtube. Pipett
on ice and mix the components by pipetting gently up and down. In
general, water, RNA and primers should be mixed together before
the remaining components are added.

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 SCRIPT Reverse Transcriptase
Reverse Transcriptase with increased thermal stability

component stock conc. final conc. 20 µl component stock conc. final conc. 20 µl
assay assay
RNase-free - - fill up to RNase-free - - fill up to
water 20 µl water 10 µl
RNA tem- - total RNA: x µl RNA tem- - total RNA: x µl
plate 10 pg - 5 µg plate 10 pg - 5 µg
or mRNA: or mRNA:
10pg-500ng 10pg-500ng
Primer 10 µM -gene- - 1-2 µl Primer 10 µM -gene- - 1-2 µl
specific - 5 µl specific - 5 µl
primer: - 5 µl primer: - 5 µl
10-20 pmol 10-20 pmol
(50-100 ng) (50-100 ng)
-oligo-dT15-25 -oligo-dT15-25
primer: primer:
50 pmol 50 pmol
(300 ng) (300 ng)
-random -random
primer: primer:
50 pmol 50 pmol
(100 ng) (100 ng)
SCRIPT RT 5x 1x 4 µl
Denaturation and primer annealing
Buffer
Incubate the mixture at 65-70 °C for 5 min and place it at room
complete
temperature (if using specific primer) or on ice (if using oligo-dT or
dNTP Mix 10 mM each 500 µM 1 µl random primer).
each
DTT stock 100 mM 5 mM 1 µl Preparation of the Reaction Mix
solution1) Add the following components to a further nuclease-free microtube
RNase 40 units/µl 20 units 0,5 µl and mix by pipetting gently up and down:
Inhibitor2) component stock conc. final conc. 20 µl
SCRIPT 200 units/µl 100-200 0.5-1 µl assay
Reverse units RNase-free - - fill up to
Transcriptase3) water 10 µl
1) Adding of up to 5 mM DTT may increase the yield and is recommen- SCRIPT RT 5x 1x 4 µl
ded for individual optimization. Buffer
2) Addition of 20-40 units RNase inhibitor per assay is recommended complete
and may be essential when working with low amounts of starting dNTP Mix 10 mM each 500 µM 1 µl
RNA. each
3) 100 units (0.5 µl) of enzyme is recommended for standard assays
DTT stock 100 mM 5 mM 1 µl
but increasing the amount of enzyme to 200 units (1 µl) per assay solution1)
may show even higher transcription yields under selected assay RNase 40 units/µl 40 units 1 µl
conditions. Inhibitor2)
1b Assay set-up with sample denaturation
(RNA/primer with a high degree of secondary structure) SCRIPT 200 units/µl 100-200 0.5-1 µl
Preparation of the RNA Template / Primer Mix Reverse units
Add the following components to a nuclease-free microtube and mix Transcriptase3)
by pipetting gently up and down: 1) Adding of up to 5 mM DTT may increase the yield and is recommen-
ded for individual optimization.
2) Addition of 20-40 units RNase inhibitor per assay is recommended

and may be essential when working with low amounts of starting

Jena Bioscience GmbH

Löbstedter Str. 71 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
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http://www.jenabioscience.com
Version: 0001, Last update: May 18, 2020
 SCRIPT Reverse Transcriptase
Reverse Transcriptase with increased thermal stability

RNA.
3) 100 units (0.5 µl) of enzyme is recommended for standard assays

but increasing the amount of enzyme to 200 units (1 µl) per assay
may show even higher transcription yields under selected assay
conditions.

Complete Reaction Mix

Add 10 µl Reaction Mix to 10 µl RNA Template / Primer Mix to
complete the 20 µl Reaction Mix. Pipett on ice and mix by pipetting
gently up and down.

2 First-strand cDNA synthesis

Incubation
Incubate the reaction mix at 50 °C for 30-60 min if using gene-specific
primers. If using oligo-dT or random primers incubate at 42 °C for
10 min followed by incubation at 50 °C for 30-60 min.
[Please note: The optimal time depends on the length of cDNA.
Incubation of 60 min is recommended for cDNA fragments of more
than 2,000 bp length. The optimal temperature depends on the
structural features of the RNA. Increase the temperature to 55 °C
for difficult templates with high secondary structure. Note that
optimal reaction time and temperature should be adjusted for each
particular RNA.]

Optional: Heat inactivation

Heat the mixture to 70 °C for 10 min to inactivate the Reverse
Transcriptase.

Optional: RNA removal

Add 2 units DNase-free RNase and incubate at 37 °C for 20 min.
The cDNA can now be used as template in PCR or be stored at -20 °C.
Apply 2-5 µl of the RT assay for further amplification in PCR.
However, some specific DNA applications may require the prior
inactivation of the remaining RTase or the enzymatic removal of RNA.

Activity:
Activity and stability tested in first strand cDNA synthesis.

Jena Bioscience GmbH

Löbstedter Str. 71 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
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Version: 0001, Last update: May 18, 2020